Hypertension is a cardiovascular risk factor that can lead to ischemic injury, myocardial infarction (Ml)and heart failure. During these disease processes, the heart undergoes hypertrophy and fibrosis, referred to as cardiac remodeling. Remodeling is stimulated by mechanical factors, release of pro-inflammatory cytokines, neurohormonal agents (p-adrenergic hormones) and vasoactive hormones with trophic properties, such as angiotensin II (Ang II). These agents also regulate the genes involved in the inflammatory response, including cyclooxygenase-2 (COX-2) and the PGE2 synthase mPGES-1, resulting in substantial production of the prostanoid PGE2 by cardiac myocytes. We have shown that COX-2 is induced in the mouse heart following Ml, and 2 wk treatment with a specific COX-2 inhibitor improves cardiac function and reduces hypertrophy and fibrosis. In vitro studies using cardiac myocytes indicate that binding of PGE2 to its receptor, EP4, results in transactivation of the epidermal growth factor receptor (EGFR), activation of p42/44 MAPK and increased protein synthesis. The gene marker of hypertrophy b-type natriuretic peptide (BMP) is also regulated by PGE2. We hypothesize that PGE2 (generated by mPGES-1) and EP4 contribute to end organ damage (cardiac hypertrophy and inflammation) in response to Ang ll-induced hypertension and ischemic injury (caused by Ml) via activation of EGFR and p42/44 MAPK. In Aim I we will study the signaling molecules involved in EGFR transactivation and downstream p42/44 MAPK activation. We will also overexpress EP4 in myocytes to study its internalization via endocytosis and the contribution of endocytosis to p42/44 MAPK signaling. In Aim II we will use transient transfection of the BMP promoter and treatment with pharmacological and molecular inhibitors of signaling molecules such as Src, small GTPases and EGFR to further elucidate EP4-dependent events in myocytes. We will also study other kinases and transcription factors downstream from p42/44 MAPK that are involved in myocyte growth, including Egr-1 and GATA-4. In Aims III and IV, we will use novel mutant mouse lines to examine how cardiac myocyte-specific overexpression and deletion of EP4 and deletion of mPGES-1 modulate hypertrophy in models of Ang ll-dependent hypertension and Ml. The contribution of the COX-2 product PGE2 and its receptor EP4 to chronic cardiac pathophysiology is virtually unexplored. Our studies will use an integrative approach to study the cellular and molecular basis for the deleterious effects of the inflammatory prostanoid PGE2 and its receptor both in vitro and in novel mouse models in vivo. Abbreviations: ACM = adult cardiac myocytes; ADAM = a disintegrin and metalloproteinase protein; Ang II = angiotensin II; p2AR = p2-adrenergic receptor; BNP = B-type natriuretic peptide; COX = cyclooxygenase; EGFR = epidermal growth factor receptor; Egr-1 = early growth response factor 1; EOD = end organ damage; EP = PGE2 receptor; EP4 = PGE2 receptor type 4; Grk = G-protein coupled receptor kinase; HB-EGF = heparin- binding epidermal growth factor; IL-1 = interleukin-1p; KO or -/- = gene knockout; LAD = left anterior descending coronary artery; LV = left ventricle; Ml = myocardial infarction; mPGES-1 = inducible membrane- localized PGE2 synthase; NVM = neonatal ventricular myocytes; p42/44 MAPK = p42/44 mitogen-activated protein kinase = Erk1/2; PGE2 = prostaglandin E2; PKA = protein kinase A; ROS = reactive oxygen species; siRNA = small interfering RNA molecule. PHS 398/2590 (Rev. 09/04, Reissued4/2006) Page 236 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): LaPointe, M.C., Ph.D/Carretero, Oscar A., M.D. A.